pharmaceutics Article Magnetite Nanoparticles Functionalized with RNases against Intracellular Infection of Pseudomonas aeruginosa Nathaly Rangel-Muñoz 1,† , Alejandra Suarez-Arnedo 1,† , Raúl Anguita 2 , Guillem Prats-Ejarque 2 , Johann F. Osma 3 , Carolina Muñoz-Camargo 1 , Ester Boix 2, * , Juan C. Cruz 1, * and Vivian A. Salazar 2,3, * 1 Department of Biomedical Engineering, Universidad de los Andes, Cra. 1E No. 19a-40, Bogotá 111711, Colombia; in.rangel@uniandes.edu.co (N.R.-M.); sa.suarez1912@uniandes.edu.co (A.S.-A.); c.munoz2016@uniandes.edu.co (C.M.-C.) 2 Department of Biochemistry and Molecular Biology, Faculty of Biosciences, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain; raul.anguita@e-campus.uab.cat (R.A.); Guillem.Prats.Ejarque@uab.cat (G.P.-E.) 3 Department of Electrical and Electronics Engineering, Universidad de los Andes, Cra. 1E No. 19a-40, Bogotá 111711, Colombia; jf.osma43@uniandes.edu.co * Correspondence: Ester.Boix@uab.es (E.B.); jc.cruz@uniandes.edu.co (J.C.C.); visalazar@uniandes.edu.co (V.A.S.) † These authors have contributed equally to this work.  Received: 4 June 2020; Accepted: 24 June 2020; Published: 6 July 2020  Abstract: Current treatments against bacterial infections have severe limitations, mainly due to the emergence of resistance to conventional antibiotics. In the specific case of Pseudomonas aeruginosa strains, they have shown a number of resistance mechanisms to counter most antibiotics. Human secretory RNases from the RNase A superfamily are proteins involved in a wide variety of biological functions, including antimicrobial activity. The objective of this work was to explore the intracellular antimicrobial action of an RNase 3/1 hybrid protein that combines RNase 1 high catalytic and RNase 3 bactericidal activities. To achieve this, we immobilized the RNase 3/1 hybrid on Polyetheramine (PEA)-modified magnetite nanoparticles (MNPs). The obtained nanobioconjugates were tested in macrophage-derived THP-1 cells infected with Pseudomonas aeruginosa PAO1. The obtained results show high antimicrobial activity of the functionalized hybrid protein (MNP-RNase 3/1) against the intracellular growth of P. aeruginosa of the functionalized hybrid protein. Moreover, the immobilization of RNase 3/1 enhances its antimicrobial and cell-penetrating activities without generating any significant cell damage. Considering the observed antibacterial activity, the immobilization of the RNase A superfamily and derived proteins represents an innovative approach for the development of new strategies using nanoparticles to deliver antimicrobials that counteract P. aeruginosa intracellular infection. Keywords: Pseudomonas aeruginosa; ribonucleases; magnetite nanoparticles; antimicrobials 1. Introduction The rapid emergence of resistant bacteria is occurring worldwide, endangering the efficacy of antibiotics and turning into a public health threat that can compromise millions of lives [1]. The antibiotic resistance crisis has been attributed to the overuse and misuse of these medications, as well as a lack of new drug development approaches against the emergence of multi-drug resistance (MDR) strains [2]. Over the last decade, studies have focused on the search and evaluation of new Pharmaceutics 2020, 12, 631; doi:10.3390/pharmaceutics12070631 www.mdpi.com/journal/pharmaceutics Pharmaceutics 2020, 12, 631 2 of 25 biomolecules, such as peptides and proteins, with potent antimicrobial activities [3,4]. These emerging molecules have been reported to act either alone or in combination to potentiate their action against pathogens. Antimicrobial proteins and peptides (AMPs) are naturally-occurring and abundantly distributed in various organs, tissues, and body fluids as part of the human innate system [5,6]. Human ribonucleases (RNases) of the superfamily of RNase A are a vertebrate-specific family of proteins of structurally homologous proteins to the bovine pancreatic ribonuclease A, also known as RNase A; this is perhaps, the best-characterized of all known mammalian enzyme proteins [7]. In humans, eight functional RNases’ (named canonical RNases [8]) genes have been identified within a single chromosome. This enzyme family includes RNase 1, or pancreatic RNase, expressed in the pancreas as well as in other organs and tissues [9]. Among all the canonical RNases, RNase 1 has the highest catalytic activity and a remarkable activity against dsRNA involved in the nonspecific response to pathogenic RNA molecules [10,11]. Regarding antimicrobial activity, RNase 3 stands out for its high bactericidal action and the efficacy of an N-terminus-derived peptide on Gram-negative bacteria biofilms. This ability to remove biofilms of Gram-negative species can be explained by its specific cell agglutination and lipopolysaccharide-binding properties [12–15]. Besides, RNase 3 was proved effective against macrophage intracellular infection. Interestingly, we have demonstrated the high performance of RNase 3, not only in bacterial infection removal but also in internalization within macrophage cells and autophagy activation [16]. In this context, we designed an RNase 3/1 hybrid that combines the unique features of RNase 3 and the high catalytic activity and ribonuclease inhibitor affinity of RNase 1 and tested the construct in an in vitro experimental evolution test with Acinetobacter baumannii, where it demonstrated its ability to delay the acquisition of colistin resistance [15]. Antimicrobial RNases have been reported to be effective against biofilm-forming pathogens, such as Mycobacterium tuberculosis, A. baumannii, or Pseudomonas aeruginosa [12,13]. P. aeruginosa is classified as an opportunistic pathogen that has been commonly associated with deadly infections, such as pneumonia. Moreover, among others, P. aeruginosa is a serious threat to immunocompromised and cystic fibrosis (CF) patients [17,18]. Some of these common health problems have been aggravated due to the emergence of bacterial resistance to drugs. For this reason, P. aeruginosa has been listed among the pathogens for the prioritized search of new antimicrobials [19]. Additionally, P. aeruginosa can be internalized by macrophages and escape vacuole entrapment. Moreover, once internalized, P. aeruginosa fosters a phase of intracellular residence mediated by several adaptation mechanisms that help the pathogen avoiding phagocytosis and ultimately, promoting extracellular multiplication and replication in other host cells [20–25]. Many studies have demonstrated the potency of cell internalization of pharmacological treatments as a very effective route to tackle persistent P. aeruginosa infections [17,20–22,25,26]. In particular, one pharmacological avenue of interest is the delivery of engineered AMPs to improve their internalization capacity. Some of the engineering strategies include functionalization with cysteine-pentaglycyl, o-dithiobenzyl carbamate, (acyloxy)alkyl ester and polyethylene glycol (PEG) [27–29]. Additionally, an emerging route to increase penetration is provided by the widely available nanocarriers. In this regard, a number of studies have been dedicated to interfacing AMPs with nanomaterials, such as iron oxide, gold, silver and carbon nanoparticles, to enhance penetration significantly and consequently, bioavailability [28,30–32]. In the case of microbial control via delivery of the nanobioconjugates of peptide drugs, recent successful examples include Cathelicidin LL-37-carbon nanoparticles, Buforin II-MNPs and Ubiquicidin (29–41)-silver-gold nanoparticles [31–35]. One of the most attractive nanodelivery systems is magnetite nanoparticles (MNPs), mainly due to their magnetic properties, which allow them to be a reliable platform for magnetic targeting, as external magnetic fields can be applied to guide their transport, fate and improve their localization within target tissues [36]. Additionally, once delivered into a particular organ or tissue, magnetic fields can be subsequently applied to facilitate processes such as thermal energy release, endosomal escape or disrupting endothelial cell–cell junctions [36]. MNPs can be easily visualized with the aid of MRI and magnetic particle imaging (MPI) instruments, which facilitate the ease monitoring of their localization within organs, tissues and cells both in in vitro and in vivo [36,37]. MNPs exhibit Pharmaceutics 2020, 12, 631 3 of 25 high biocompatibility and antimicrobial abilities [33,34,38]. They also have been widely employed to immobilize enzymes, mainly due to the possibility of enhancing their catalytic performance [39], thermal and long term stability [40] and reusability [41–43].We recently developed cell-penetrating nanobioconjugates of MNPs by interfacing them with the AMP Buforin II (BUF-II) and the Outer Membrane Protein A (OmpA) of Escherichia coli [33,34]. The synthesized nanobioconjugates not only penetrated a number of mammalian cell lines but efficiently escaped endosomes. Moreover, they exhibited high biocompatibility, as evidenced by low cytotoxicity and reduced hemolytic tendency. To maintain the antimicrobial activity of BUF-II, we modified the MNPs with a polyetheramine (PEA) surface spacer. The polyetheramines contain oxygen moieties that are described in the literature as highly biocompatible. Additionally, these spacers have been reported as useful surface linkers because of the flexibility imparted by the polyether groups present in their backbones [44]. Besides, it has been reported that RNases have been immobilized on different types of supports, including magnetic microspheres [45], dextran nanogels [46], silica nanoparticles [47], and lipidoids [48]. The prepared immobilizates found application in the delivery of RNase A for cancer treatment [49] and RNase H for the detection of pathogenic bacterial DNA [50]. By taking advantage of the potent antimicrobial activity reported for hybrid RNase 3/1 [15], we designed the bionanoconjugate MNP-RNase 3/1. This work is therefore dedicated to immobilizing the hybrid RNase 3/1 on magnetite nanoparticles to increase therapeutic efficacy against P. aeruginosa at the intracellular level. We synthesized and functionalized the nanoparticles to obtain the RNase 3/1 or RNase A nanobioconjugates. Then, they were characterized with the aid of light scattering, spectroscopy, and thermal techniques. Moreover, we evaluated their biocompatibility, cytotoxicity, cell-penetrating abilities, and the control of extracellular and intracellular infection by P. aeruginosa. 2. Materials and Methods 2.1. Materials Magnetic nanoparticle (MNP) synthesis and functionalization were conducted with 98% Iron (II) chloride tetrahydrate (PanReac AppliChem, Chicago, IL, USA) and 97% Iron (III) chloride tetrahydrate from (Sigma-Aldrich Co., Munich, Germany), 98% sodium hydroxide (NaOH), dimethylformamide (DMF) (PanReac AppliChem, Chicago, IL, USA), 25% tetramethylammonium hydroxide (TMAH), 98% (3-Aminopropyl) triethoxysilane (APTES), 98% N-hydroxysuccinimide (NHS), 98% N-[3-dimethylammino)-propyl]-N0 -ethyl carbodiimide hydro-chloride (EDC), 99% dimethyl sulfoxide (DMSO), >95% Rhodamine B, and 25% glutaraldehyde (Sigma-Aldrich, Munich, Germany). Triton™ X-100 was purchased from (Sigma-Aldrich). Ribonucleic acid from baker’s yeast (S. cerevisiae) (Sigma-Aldrich, Munich, Germany) was assayed for catalytic analysis. Luria–Bertani (LB) for P. aeruginosa PAO1 (Sigma Aldrich, Munich, Germany) were used for the bacterial cultures. Jeffamine M-600 polyetheramine (PEA) (Huntsman, Salt Lake City, UT, USA) was used as a surface spacer and oxidized with the aid of KMnO4 (Sigma-Aldrich, Munich, Germany) to render free terminal carboxyl groups for further conjugation. DMEM, RPMI 1640 Medium, fetal bovine serum (FBS) and trypsin EDTA were obtained from Biowest (Barcelona, Spain). Penicillin/Streptomycin (P/S) was purchased from Lonza (Basel, Switzerland) and Gentamicin (80 mg/mL) were purchased from Genfar (Bogotá, Colombia). Phorbol 12-myristate 13-acetate (PMA) was used to induced monocyte THP-1 differentiation into macrophages (Santa Cruz Biotechnology, Dallas, TX, USA). Cytotoxicity and delivery were carried out in THP-1 (NCTC, #88081201) and Vero (ATCC® CCL-81). Cell dyes—Vybrant™ DiO Cell-Labeling Solution, LysoTracker™ Green DND-26 and Hoechst 33342 Staining Dye Solution—were all purchased from Invitrogen (Carlsbad, CA, USA). Finally, the bacterium strain was P. aeruginosa (ATCC 15915). Pharmaceutics 2020, 12, 631 4 of 25 2.2. Synthesis of Magnetic Nanoparticles A mixture of 1 g of FeCl2 ·4H2 O and 2.71 g of FeCl3 ·6H2 O was dissolved separately in 5 mL of distilled water to obtain 1 and 2 M solutions, respectively. Chloride solutions were then mixed and heated up until a homogeneous solution was obtained. When the solution reached 90 ◦ C, 2 mL of 2% v/v solution of TMAH was added. At the same time, 1.6 g of NaOH were dissolved in 5 mL of Type I water (8 M solution), and this solution was also heated to 90 ◦ C. The hot NaOH solution was finally dropped at 200 µL/min into the chloride mixture under vigorous stirring at 1500 rpm. The reaction was carried out for 1 h at 90 ◦ C under continuous stirring at 1500 rpm. The obtained MNPs were washed at least 3 times with distilled water with the aid of a strong permanent magnet, and sonicated After each wash (2800 ultrasonic cleaner, Branson, MO, USA) for 15 min at 40 kHz frequency. The obtained MNPs size was measured via Dynamic Light Scattering (DLS) (Zeta-Sizer Nano-ZS, Malvern, UK). 2.3. Surface Functionalization For surface functionalization, 100 mg of magnetite were dissolved in distilled water and sonicated vigorously until complete homogenization. In total, 2 mL of 2% (v/v) solution of TMAH, 50 µL of 99% glacial acetic acid and 100 µL of 10% (v/v) APTES were added to resuspend the MNPs. The sample was then kept for 1 h at 60 ◦ C and 250 rpm to carry out the chemical reaction. Finally, the sample was washed several times with 1.5% (w/v) NaCl saline solution and Type I water to remove excess reagents. 2.4. Ribonuclease A and Ribonuclease 3/1 Immobilization An amount of 2 mL of 2.5% (v/v) solution of glutaraldehyde was added to 100 mg of the MNPs sample and the mixture was left under mechanical agitation at 250 rpm at room temperature. Next, 300 µL of oxidized PEA were added and left overnight at 250 rpm. Then, 25 mg of RNase protein diluted in distilled water was mixed with an aqueous solution of 5 mg of NHS and 5 mg of EDC as crosslinking agents at 30 ◦ C. The sample was left at 250 rpm overnight to obtain the MNPs-RNase nanobioconjugates. Finally, the MNPs-RNase samples were washed with NaCl solution 1.5% (w/v) and distilled water to remove excess reagents, aided by a permanent magnet. The effective conjugation of the RNase on the MNPs was verified with the aid of Fourier-transform Infrared Spectroscopy (FTIR) (Bruker Alpha II FTIR Eco-ATR (Bruker, Ettlinglen, Germany) and Thermogravimetric Analysis (TGA) (TA Instruments, New Castle, DE, USA). The concentration of immobilized protein per mass of nanoparticles was quantified colorimetrically using a commercial BCA Protein Assay Kit [51] (Supplementary Materials Figure S1) (Sigma-Aldrich, Munich, Germany). 2.5. Labeling of MNPs-RNase Nanobioconjugates with Rhodamine B A mixture of 5 mg of EDC and 5 mg of NHS was mixed with 5 mL of distilled water under vigorous agitation at 210 rpm and 38 ◦ C temperature for 10 min. Next, 3 mg of Rhodamine B was added to the mixture and vortexed to obtain a homogeneous solution. Finally, the Rhodamine B solution was added to 10 mg of MNPs-RNase covered with aluminum foil to protect from photobleaching and left under agitation and refrigerated overnight. The labeled sample was washed several times under sterile conditions with distilled water until no traces of Rhodamine B were detectable. 2.6. Hemolysis Assay Hemolytic activity of MNPs-RNase was quantified according to Cuellar et al. [33]. A blood sample was obtained from a healthy volunteer in vacutainer blood tubes with EDTA. The samples were obtained with the approval of the Ethical Committee at the Universidad de los Andes (minute number 928-2018). In total, 25 mL of blood was centrifuged at 1800 rpm for 5 min; After that, the plasma supernatant was removed and replaced with 0.9% (w/v) NaCl solution. This process was performed in triplicate and the last supernatant was replaced by PBS buffer at pH 7.4. Serial dilutions of MNPs and MNPs-RNase were prepared in concentrations from 100 µg/mL to 12.5 µg/mL. In total, 100 µL of the Pharmaceutics 2020, 12, 631 5 of 25 MNPs-RNase were mixed with 100 µL of diluted 1:10 red blood cells and incubated in a 96-well plate for 1 h at 37 ◦ C. Triton X-100 and PBS 1× were used as positive and negative controls, respectively. 2.7. Platelet Aggregation Test As for the hemolysis assay, a blood sample from a healthy volunteer was obtained with vacuum blue collection tubes containing Na3 C6 H5 O7 and centrifuged at 1000 rpm for 15 min until a transparent supernatant with platelets was obtained. Samples of MNPs, MNPs-RNase A and MNPs-RNase 3/1 were previously sonicated and suspended in PBS 10× solution and finally, transferred to a microplate well to form serial dilutions from 100 to 6.25 µg/mL in triplicate. A blank without any nanomaterials was included in the experimental set. Platelets were added to each sample and left to interact for 3 min at room temperature. Subsequently, samples were measured in a microplate spectrophotometer (Thermo Scientific™, Waltham, MA, USA) at 620 nm. Thrombin and epinephrine were used as high positive platelet aggregation controls and PBS as the negative control. 2.8. LDH Cytotoxicity Assay The MNPs-RNase cytotoxicity was carried out on human monocytic THP-1 and African green monkey kidney Vero cells via lactate dehydrogenase (LDH) assay in 96-well cell culture flat-bottom plates [21]. Nanobioconjugates of MNPs-RNase A and 3/1 were serially diluted in RPMI or DMEM medium from 1 to 0.3 mg/mL and then, added to a cell density of 5 × 105 cells/mL. Samples were incubated for 24 h. The positive control was cells treated with 10% Triton X-100, while the negative control was untreated cells. Then, samples were centrifuged at 1100 rpm for 5 min and 50 µL of supernatant was transferred to a 96-well plate, mixed with 50 µL of LDH reaction mixture and incubated for 30 min. Finally, absorbance was measured at 490 and 650 nm for reference. 2.9. Catalytic Evaluation of MNPs-RNases by Activity Staining Plates To evaluate the catalytic activity of RNases After immobilization, the samples were analyzed by staining activity agarose plates with total RNA yeast used as substrate by following the protocol of Radomski et al. [24]. MNPs-RNases and protein samples were placed over a plate that contained 0.1% of yeast RNA, 1% agarose in 20 mM sodium acetate (pH 5.0) at 37 ◦ C for 30 min. Proteins were assayed at a 400 ng concentration and MNPs-RNase at 5000 ng. The percentage of catalytic activity was measured as a ratio between the bright area and the dark background using ImageJ/Fiji® (Madison, WI, USA). 2.10. Bacterial Cell Viability Luminescent Assay Bacterial viability was assayed using the BacTiter-Glo™ microbial cell viability kit, which allows measuring the viability of cells by ATP quantification with an indirectly measured luminescence detection assay. Overnight bacterial culture was subcultured until it reached OD600 = 0.4. Subsequently, serial dilutions of ribonucleases and nanobioconjugates (from 500 to 15 µg/mL) were incubated with P. aeruginosa PAO1 (1.6 × 106 cells) in 10 mM HEPES buffer (pH 7.5) for 4 h at 37 ◦ C. After incubation, 50 µL of the BacTiter-Glo™ reagent was added to each well and incubated at room temperature for 15 min. Luminescence was read on a Victor3 plate reader (Perkin-Elmer, Waltham, MA, USA) with a 3 s integration time. Luminescence signal to viable cell counts correlation was checked for the untreated P. aeruginosa cell controls. MBC was defined as the lowest concentration of MNPs, protein or MNPs-protein that resulted of >90% reduction in the initial inoculum. 2.11. THP-1 Cell Culture and Macrophage Induction Human THP-1 cell culture was maintained at 37 ◦ C in 25 cm2 tissue culture flasks using RPMI-1640 medium with 10% FBS and 1% P/S. Cell viability was assessed by Trypan Blue counting. THP-1 cells were treated with phorbol 12-myristate 13-acetate (PMA; 100 nM; (Santa Cruz Biotechnology, Dallas, Pharmaceutics 2020, 12, 631 6 of 25 TX, USA) at 37 ◦ C for at least 24 h to obtain PMA-differentiated THP-1 macrophages. They were then cultured in RPMI-1640 at 37 ◦ C in a humidified 5% CO2 incubator. Cells were allowed to rest for 24 h before infection and exposure to treatments. 2.12. Macrophage Infection P. aeruginosa PAO1 was cultured at 37 ◦ C in a LB bacterial broth overnight. Prior to THP-1 cell infection, bacterial cells were incubated in fresh LB medium to an OD600 of 0.2, as previously established [52]. Cell culture concentration was adjusted to 3 × 105 cells/mL and infected with P. aeruginosa at a MOI of 10 resuspended in RPMI-1640 medium in 96-well plates. Time of infection was 3 h followed by 3 washes with fresh PBS. Gentamicin at 400 µg/mL was added to the cell culture to remove the presence of residual extracellular P. aeruginosa. Finally, infected macrophages were washed and treated by MNPs-RNase A and MNPs-RNase 3/1 treatment in concentrations ranking from 500 to 7 µg/mL for 24 h. After the treatment exposure, cells were lysed with distillated water and seeded in a Luria–Bertani agar plate. Colony-forming units (CFU) were counted After 24 h to calculate the Intracellular minimum Inhibitory Concentration (IMIC). 2.13. Cell Translocation of Nanobioconjugates Cell translocation of MNPs-RNase A and MNPs-RNase 3/1 was performed on monocytic cell line derived from acute monocytic leukemia (THP-1) and induced macrophages. MNPs-RNase were previously labeled with Rhodamine B for observation under the confocal microscope. First, THP-1 cells were incubated with each MNPs-RNase A or MNPs-RNase 3/1 treatment at the intracellular MIC concentration of the MNPs-RNase 3/1 for 1 h. Then, the cells were washed twice with PBS 1× to prevent the interference of the non-penetrating bionanoconjugates. RPMI was prepared with Hoechst 33342 (1:1000 dilution) and Lysotracker (1:10,000 dilution) and then, 1 mL was added to cells. Samples were later imaged under a confocal laser scanning microscope (CLSM) with a PlanApo 60×, 1.35 NA oil-immersion objective with Ex/Em 358/461 nm (Hoechst 33342), Ex/Em 504/511 nm (Lysotracker green), and Ex/Em 577-556/590-580 (Rhodamine B). Images were collected at different positions throughout the culture depth to ensure a plot z and y-stack scan. For each z-stack image of THP-1 cells, we collected about 20 images along the depth of the region of interest. ImageJ/Fiji® were used for image analysis. 2.14. Internalization Percent Determination Internalization percentage in THP-1 cells was calculated by recording the fluorescent intensity of labeled nanobioconjugates of MNPs-RNase A and MNPs-3/1 After 1 h of exposure. This was accomplished by seeding 3 × 104 cells/well in 96-well black plates followed by exposure to 100, 50, 25, 12.5, and 6.25 µg/mL of the labeled nanobioconjugates suspended in phenol-red-free RPMI medium. Fluorescence intensity of labeled nanobioconjugates was recorded in a spectrofluorometer (FluoroMax-4, Horiba, Japan) with Ex/Em wavelengths at 523/625 nm, respectively. Autofluorescence of cells and unlabeled nanobioconjugates were used as blanks. 2.15. Infection of Macrophages with Stained P. aeruginosa We prepared macrophages from THP-1 cells, as described above. Prior to infection, we stained P. aeruginosa cells with 1:200 concentration of DIO dye to facilitate their intracellular observation [53]. Macrophages were infected as described previously and left for 25 min to allow P. aeruginosa phagocytosis [21]. After exposure, macrophages were washed with PBS and we added 1:1000 ratio of Hoechst 33342 in RPMI supplemented with 400 µg/mL of gentamicin for nuclei staining. Macrophages were also exposed to labeled nanobioconjugates of MNPs-RNase A and MNPs-RNase 3/1 at the intracellular MIC (IMIC) for 1 h. Finally, the cells were washed with PBS before observation by confocal microscopy at the corresponding Ex/Em wavelengths for stained nuclei and nanobioconjugates (see above). In the case of labeled P. aeruginosa, imaging was conducted at Ex/Em484/501 nm. Pharmaceutics 2020, 12, 631 7 of 25 2.16. Statistical Analysis The Student’s t-test and two-way ANOVA were applied to evaluate the significance of the differences between treatments. The results were plotted and analyzed using OriginPro 2018® (OriginLab Corporation, Wellesley, MA, USA). 3. Results 3.1. Ribonuclease A and Ribonuclease 3/1 Immobilization on Magnetite Nanoparticles (MNPs) Recently, we prepared bionanoconjugates of MNPs interfaced with antimicrobial and translocating molecules [33,34]. Here, we followed the immobilization protocol that led to superior antibacterial activity for the AMP Buforin II. According to this approach, immobilization proceeded on MNPs modified with an oxidized PEA surface spacer [33]. Consequently, we conducted immobilization of RNase 3/1 and RNase A on PEA modified MNPs to preserve their biological function. The bionanoconjugate obtained After immobilization of RNases on PEA-modified MNPs is shown schematically in Figure 1A. Figure 1B shows the hydrodynamic diameter (HD) distribution of bare MNPs in contrast with the MNP-RNases bionanoconjugates as determined by DLS. Initially, bare MNPs approached a mean hydrodynamic diameter of 91 nm with a polydispersity index (PI) of 49%. After PEA coating, HD was measured at 460 nm with a %PI of 23 and finally, After conjugation of RNase 3/1, the hydrodynamic diameter increased to 290 nm with a PI of 22%. Those values of HD and PI% for MNP prior and After immobilization are comparable to those obtained in our previous work [33]. As shown in Figure 1B, MNP-RNase presents higher hydrodynamic diameters, thereby reproducing our previous results After conjugation of Buforin-II on PEA-modified MNPs [33]. The increase in hydrodynamic diameter is most likely due to aggregation phenomena during the immobilization of RNases [54]. This undesired effect most likely takes place during the washing steps, where 1.5% NaCl is added to the suspension to neutralize superficial charges and induce precipitation of the nanobioconjugates. Immobilization is also likely to expose hydrophobic moieties of the proteins, which exhibit a marked tendency for interaction and ultimately, promote clustering. To overcome this issue, it was necessary to perform multiple washes with ultrapure water and prolonged sonication periods. Immobilization of both RNases was verified by FTIR and TGA analyses. Figure 1C shows the spectra for bare magnetite, MNPs-RNase A and MNPs-RNase 3/1 bionanoconjugates. In the case of bare magnetite, the spectrum exhibited an absorption band at around 564 cm−1 , which can be attributed to the Fe-O bond of iron oxide and is present in all conjugation steps [55]. Observation of the Si-O-Si stretching vibration at about 1006 cm−1 confirms the presence of APTES in the MNP–AP complex. Conjugation of PEA on silanized magnetite (MNP-AP-PEA) was confirmed by an absorption peak at 1670 cm−1 for the presence of C=O stretching in the backbone of the polymer [56]. Finally, conjugation of the RNases was verified by the presence of the amide I band at 1674 cm−1 and the amide II band at 1531 cm−1 , which can be related to the secondary structure of the proteins [57]. Conjugation efficiency of RNase A and RNase 3/1 on the surface of MNP was estimated via TGA (Figure 1D). MNP and MNP-RNases exhibited an initial weight loss of about 2.0%, mainly due to dehydration of the samples. Bare magnetite had a second weight loss of 4.7%, while the MNPs-RNase A and MNPs-RNase 3/1 nanobioconjugates were 7.7% and 6.3%, respectively. These losses are attributed to the physically adsorbed organic compound residues derived from the synthesis and functionalization processes. Finally, the detachment of the RNases from the magnetite was estimated with a final weight loss step of 5.2% for MNPs-RNase A and close to 8.0% for MNPs-RNase 3/1. These weight losses agree well with those found in our previous works [33,34]. Further evidence of the conjugation efficiencies was provided by a BCA test (Figure S1), where the weight ratio between the MNPs-RNase bionanoconjugate and the free RNase protein was about 9:2 (2.277 µg per mg of MNP) for the RNase 3/1 and 4:1 (i.e., 2.319 µg per mg of MNP) for RNase A. This allowed us to estimate that 1 mg/mL of nanobioconjugates correspond to 15.52 µM for RNase A and 16.62 µM for RNase 3/1, respectively. diameter is most likely due to aggregation phenomena during the immobilization of RNases [54]. This undesired effect most likely takes place during the washing steps, where 1.5% NaCl is added to the suspension to neutralize superficial charges and induce precipitation of the nanobioconjugates. Immobilization is also likely to expose hydrophobic moieties of the proteins, which exhibit a marked tendency for Pharmaceutics interaction 2020, 12, 631 and ultimately, promote clustering. To overcome this issue, it was necessary to 8 of 25 perform multiple washes with ultrapure water and prolonged sonication periods. Figure (A) Schematic Figure 1. (A) Schematicrepresentation representationofofthe thechemical chemical structure structure of of thethe MNP-RNase MNP-RNase bionanoconjugate. bionanoconjugate. (B) (B) DLS DLS histogram histogram for for the the sizesize distribution distribution of magnetite of magnetite nanoparticles nanoparticles before before (gray), (gray), PEAPEA coated coated NPs NPs (blue) (blue) and After and after immobilization immobilization MNP-RNase MNP-RNase (red) nanobioconjugates. (red) nanobioconjugates. (C) FTIR (C) FTIRofspectra spectra of bare bare magnetite magnetite (gray), MNP-RNase A nanobioconjugates (red), and magnetite-RNase 3/1 nanobioconjugates (gray), MNP-RNase A nanobioconjugates (red), and magnetite-RNase 3/1 nanobioconjugates (blue). (blue). AI—Amide I and AII—Amide II. (D) TGA thermograms of magnetite (gray) and MNP-RNase A nanobioconjugates (red), and magnetite-RNase 3/1 nanobioconjugates (blue). The first weight loss steps (2.0%) represent the dehydration of the samples. Second weight loss steps (4.7%, 7.7%, and 6.3%) correspond to physically adsorbed organic solvents. The final weight loss steps (5.2% and 8.0%) are attributed to the detachment of RNase A and RNase 3/1 from the surface of magnetite nanoparticles. 3.2. Catalytic Activity Evaluation of MNPs-RNases The two obtained nanobioconjugates showed reduced catalytic activity when compared with the free proteins. This was evidenced After incubation for 30 min of 5 µg of MNPs RNases and 400 ng of native proteins (ratio 1:12 protein: bionanoconjugate; micromolar concentration of immobilized protein 1.16 and 1.14 for MNPs-RNase A and MNPs-RNase 3/1, respectively) on agarose enriched in yeast RNA. A quantitative analysis of Figure 2A,B shows that, when compared with the free proteins, the catalytic activity After immobilization was reduced by about 85.5% and 60.8% for RNase A and RNase 3/1, respectively (Figure 2C). We hypothesize that the loss of catalytic activity upon immobilization might be due to surface-induced conformational changes responsible for detrimentally altering the active sites, as reported elsewhere [58]. This notion should be verified by looking at possible secondary and tertiary structural changes with the aid of high-resolution biophysical techniques. Pharmaceutics 2020, 12, 631 9 of 25 Figure 2. (A) Catalytic activity toward the total RNase of yeast. Light areas correspond to the sites of Figure 2 inoculation of the samples. The assay was conducted by depositing 5 µg of MNPs RNases (1.159 µM or MNPs-RNase A and 1.138 µM for MNPs-RNase 3/1) and 400 ng of free proteins and subsequently, incubating for 30 min. Images were collected with the aid of the Quantity One software® (Bio-Rad). (B) Densitometric analysis of the light areas for native and immobilized proteins using ImageJ/Fiji® . (C) Activity ratio of nanobioconjugates compared to free proteins and activity ratio of immobilized compared to free proteins. The activity ratio between immobilized and free proteins was normalized by the amount of protein estimated via BCA. 3.3. Biocompatibility Assessment To determine the potential as an antimicrobial treatment, the obtained magnetite-RNase nanobioconjugates need to be biocompatible. For this purpose, we followed the ISO 10993 standard to evaluate their hemolytic, platelet aggregation, and cytotoxic effect. Figure 3A shows hemolysis levels remained below 5% for all concentrations tested (100 to 6.26 µg/mL), thereby complying with the ISO standard. Figure 3B shows that for concentrations of 25 µg/mL and below, MNPs and both MNPs-RNases exhibited a low platelet aggregation tendency, as evidenced by levels below those of PBS, used as a negative control. For 50 and 100 µg/mL, the aggregation levels approached those of thrombin but were only half of those obtained with epinephrine, which is a strong aggregating agent. When compared with bare nanoparticles, the presence of the immobilized RNases appears not to have a further impact on aggregation. The intermediate levels of aggregation found here for the nanobioconjugates have been reported previously for other nanomaterials [59,60]. Of initial concern for this study was the potential cytotoxicity of RNases [61]. However, recent studies demonstrated that the modified RNase 3/1 protein showed low cytotoxicity for concentrations up to 200 µM [15]. Cytotoxicity assays were conducted for two model cell lines, i.e., THP-1 and Vero. The THP-1 monocytes were used instead of macrophages due to the possible interference of the differentiating PMA (phorbol myristate acetate) molecule with the LDH reagents. As a result, the presence of this compound could potentially lead to biased results [62–64]. Figure 3C,D show cell viability results for the MNPs-RNases on THP-1 and Vero cell lines, respectively. Our findings confirm that even at the highest concentration 1 Pharmaceutics 2020, 12, 631 10 of 25 evaluated (i.e., 100 µg/mL), cell viability for both MNPs-RNase nanobioconjugates remains above 90% After 24 h. Recent studies have reported similar results for RNase A immobilized on different materials at concentrations lower than those evaluated here [46,65]. Taken together, these results suggest that the obtained nanobioconjugates are highly biocompatible, and therefore, have the potential to be considered for further in vivo studies. Figure 3. MNPs and both MNP-RNases exhibit high biocompatibility. (A) Assessment of the hemolytic Figure 3 effect of MNPs and MNPs-RNase nanobioconjugates. In all cases, hemolysis was below 5%, therefore complying with the ISO 10993 standard. Triton X-100 was used as a positive control. (B) Platelet aggregation for MNPs and MNPs-RNase nanobioconjugates. Platelet aggregation caused by MNPs and MNPs-RNase nanobioconjugates compared to Epinephrine and Thrombin. PBS was used as the negative control. For concentrations of 25 µg/mL and below, MNPs and both MNPs-RNases exhibited a low platelet aggregation tendency. (C) Cytotoxicity of MNPs and MNPs-RNase nanobioconjugates on THP-1 cells and (D) Vero cells. 3.4. Antibacterial Activity of MNPs-RNases Toward Extracellular and Intracellular P. aeruginosa We evaluated the antimicrobial efficiency of free and immobilized proteins. Antibacterial activity was determined by monitoring the cell viability of P. aeruginosa using the BacTiter-Glo™ luminescent kit. Using the coupled luciferin/luciferase assay, we quantified the ATP levels to estimate the number of viable bacterial cells in the culture. Free proteins and nanobioconjugates were assayed by serial dilutions starting from an initial concentration of 500 µg/mL. Viability of P. aeruginosa was established by measuring ATP and kanamycin was used as a positive control. The results indicated that bacteria viability decreased as a function of the bionanoconjugate concentration. As a remarkable finding, we detected antimicrobial activity when the bacterial cells were exposed to bare MNPs (Figure 4A), supporting similar studies [66]. Furthermore, free proteins exhibited an observable antimicrobial Pharmaceutics 2020, 12, 631 11 of 25 activity with a MBC90 = 31 µg/mL for RNase 3/1 (Figure 4B) and no significant inhibitory activity for RNase A (data not shown). Nonetheless, the activity was below that observed for the MNPs-RNase nanobioconjugates, which showed a high antimicrobial activity against P. aeruginosa (Figure 4C,D). In contrast, concentration of MNPs-RNase 3/1 of 15 µg/mL led to a significant reduction in viability of P. aeruginosa of ≥ 90% (10% of bacterial viability indicated by the dashed line). For MNPs-RNase A, a reduction in bacterial viability up to 90% was achieved at 125 µg/mL, as shown in Figure 4C. This result is somewhat surprising as RNase A is a direct ortholog of human RNase 1 that exhibits no antibacterial activity by itself [67–69]. These unexpected results must be likely related to the effect of MNPs in2020, Pharmaceutics nanobioconjugates, 12 as indicated above. Nevertheless, the antimicrobial effect of MNPs 11 of 25 and MNP-RNase A was much lower in comparison to the results achieved by the MNP-RNase 3/1 achieved by the MNP-RNase bionanoconjugate (which showed3/1 bionanoconjugate a reduction by more(which thanshowed 90% inabacterial reduction by more viability atthan 90% in 15 µg/mL). bacterial These viability results at 15 µg/mL). demonstrated thatThese results demonstrated immobilization of RNases onthat immobilization MNPs, of RNases and in particular on RNase of the MNPs, andhybrid, 3/1 in particular of the strategy is a suitable RNase 3/1to hybrid, reduce P.isaeruginosa a suitable viability. strategy to reduce P. aeruginosa viability. A B 100 100 P. aeruginosa viability (%) P. aeruginosa viability (%) 50 50 10 10 0 0 +Control 15 31 62 125 250 500 +Control 15 31 62 125 250 500 C MNPs concentration (mg/mL) D RNase 3/1 concentration (mg/mL) P. aeruginosa viability (%) P. aeruginosa viability (%) 100 100 50 50 10 10 0 0 +Control 15 31 62 125 250 500 +Control 15 31 62 125 250 500 MNP-RNase A concentration (mg/mL) MNP-RNase 3/1 concentration (mg/mL) Figure 4. Figure 4. Antibacterial Antibacterial activity activity of of free free RNase RNase 3/13/1 and and nanobioconjugates nanobioconjugates of of RNase RNase 3/13/1 and and A A against against P.P.aeruginosa. aeruginosa.Each Each sample sample waswas analyzed analyzed after After 4 incubation 4 h of h of incubation in 10 in 10 mM mM HEPES HEPES at pH 7.4. atThe pH negative 7.4. The negative control wascontrol was buffer. untreated untreated buffer. (A) Antibacterial (A) Antibacterial activity activity of bare MNPsoffor bare MNPs for concentrations concentrations ranging from ranging 15 to 500 from µg/mL.15 (B) to 500 µg/mL. (B) Antibacterial Antibacterial activity activity of free of free Ribonuclease 3/1Ribonuclease 3/1 forranging for concentrations concentrations from 15 to 500 µg/mL. ranging from (C) 15 toAntibacterial 500 µg/mL.activity MNP-RNase (C) Antibacterial A andMNP-RNase activity (D) MNP-RNase A and3/1(D) nanobioconjugates MNP-RNase 3/1 for concentrations ranging nanobioconjugates from 15 toranging for concentrations 500 µg/mL. fromThe15 tohorizontal 500 µg/mL. black The dashed lineblack horizontal represents dashed90%line bacterial represents viability. 90% bacterial viability. Furthermore, Furthermore, although although there there is is only 22.77 ±± 0.01 only 22.77 0.01 (%weight) (%weight) of of RNase RNase 3/1 3/1 immobilized immobilized on on the the nanoparticles, nanoparticles, the the obtained obtained antibacterial antibacterial activity activity is isbetter betterthan thanthat thatof offree freeribonuclease. ribonuclease. This This represents represents an an important important advantage advantage from from the the profit profit potential potential viewpoint, viewpoint, as as less less of of the the bioactive bioactive component component is is required required for the same product performance. These results are consistent with findings reported for the same product performance. These results are consistent with findings reported inin some some previous previous studies, studies, according according toto which, which, magnetite magnetite nanoparticles nanoparticles exhibit exhibit antimicrobial antimicrobial activity activity on on their own [70,71] or are capable of improving that of the conjugated cargo their own [70,71] or are capable of improving that of the conjugated cargo [66,72]. [66,72]. Once Once thetheextracellular extracellular activity activity of nanobioconjugates of nanobioconjugates was determined, was determined, we analyzed we their analyzed their intracellular intracellular activity in macrophages infected with P. aeruginosa. Figure 5 shows the intracellular activity in macrophages infected with P. aeruginosa. Figure 5 shows the intracellular antimicrobial activity of nanobioconjugates on infected macrophages. The data were normalized with respect to the positive control, which consisted of infected cells without exposure to nanobioconjugates. At concentrations of 62.5 µg/mL of the MNP-RNase3/1, we achieved a reduction in P. aeruginosa growth above 90%. In contrast, we needed a concentration above 500 µg/mL for both MNPs and MNP-RNase A to reduce completely the intracellular growth of P. aeruginosa. Figure 5D demonstrates the potent antimicrobial activity effect Pharmaceutics 2020, 12, 631 12 of 25 antimicrobial activity of nanobioconjugates on infected macrophages. The data were normalized with respect to the positive control, which consisted of infected cells without exposure to nanobioconjugates. At concentrations of 62.5 µg/mL of the MNP-RNase3/1, we achieved a reduction in P. aeruginosa growth above 90%. In contrast, we needed a concentration above 500 µg/mL for both MNPs and MNP-RNase A to reduce completely the intracellular growth of P. aeruginosa. Figure 5D demonstrates the potent antimicrobial activity effect of MNP-RNase 3/1 compared with bare MNPs and MNP-RNase A for concentrations above 62 µg/mL, which we considered the effective treatment concentration. A statistically significant difference was even detected for MNP-RNase 3/1 compared with bare MNPs, at a concentration of 125 µg/mL. In contrast to extracellular antimicrobial activity, the same level of intracellular reduction observed at a concentration of 62 µg/mL of bionanoconjugate (1.04 µM of total immobilized protein) required a higher concentration of the MNP-RNase 3/1 nanobioconjugates. Similar results were also obtained with MNPs-RNase A nanobioconjugates and MNPs at concentrations above 250 µg/mL. Pharmaceutics 2020, 12 12 of 25 A B P. aeruginose viability (%) 100 P. aeruginose viability (%) 100 80 80 60 60 40 40 20 20 0 0 +Control 15 31 62 125 250 500 +Control 15 31 62 125 250 500 MNPs concentration (mg/mL) MNP-RNase A concentration (mg/mL) C D MNP 100 MNP-RNase A P. aeruginosa viability (%) P. aeruginose viability (%) 100 MNP-RNase 3/1 80 80 60 * 60 40 40 20 20 0 0 +Control 15 31 62 125 250 500 15 31 62 125 250 500 MNP-RNase 3/1 concentration (mg/mL) Treatment concentration (mg/mL) Minimum intracellular Figure 5. Minimum intracellular inhibitory inhibitory concentration concentration against P. aeruginosa against P. aeruginosa inin differentiated macrophages of macrophages of (A) MNPs, (B) (B) MNPs-RNase MNPs-RNase A A and and (C) (C) MNPs-RNase MNPs-RNase 3/1 3/1 nanobioconjugates. nanobioconjugates. THP-1cells Differentiated THP-1 cellswere were exposed exposed at aatmultiplicity a multiplicity of infection of infection (MOI)(MOI) of 10:1.of 10:1. Posteriorly, Posteriorly, the cells the cells were were incubated incubated for 3h, for 3h, washed andwashed maintainedand in maintained in a fresh a fresh medium medium with supplemented supplemented gentamicin.with (D) gentamicin.differences Significant (D) Significant differences between between MNPs and MNPs and MNP-RNase MNP-RNase A or MNP-RNase A or 3/1MNP-RNase were estimated3/1 were with estimated the two-waywith the two-way ANOVA ANOVA and Dunnett and*Dunnett tests. 0.05.* p-value < 0.05. p-value